A general modular approach for the solubility tagging of BODIPY dyes

Article abstract of DOI:10.1016/j.dyepig.2019.107545

We describe a general and practical strategy for the direct one-step incorporation of a tunable solubility module at the boron atom of F-BODIPY dyes. The tethering reaction uses easy-to-handle reagents, has broad functional group compatibility and proceed

Full text of DOI:10.1016/j.dyepig.2019.107545

Accepted Manuscript
A general modular approach for the solubility tagging of BODIPY dyes
Alberto Blázquez-Moraleja, Delia Álvarez-Fernández, Ruth Prieto Montero,
Inmaculada García-Moreno, Virginia Martínez-Martínez, Jorge Bañuelos Prieto, Inés
Sáenz-de-Santa-María, María D. Chiara, Jose Luis Chiara
PII:
S0143-7208(19)30687-4
DOI:
https://doi.org/10.1016/j.dyepig.2019.107545
Article Number: 107545
Reference:
DYPI 107545
To appear in: Dyes and Pigments
Received Date: 28 March 2019
Revised Date: 1 May 2019
Accepted Date: 8 May 2019
Please cite this article as: Blázquez-Moraleja A, Álvarez-Fernández D, Montero RP, García-Moreno I,
Martínez-Martínez V, Bañuelos Prieto J, Sáenz-de-Santa-María Iné, Chiara MaríD, Chiara JL, A general
modular approach for the solubility tagging of BODIPY dyes, Dyes and Pigments (2019), doi: https://
doi.org/10.1016/j.dyepig.2019.107545.
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ACCEPTED MANUSCRIPT
“Click-type” solubility-tagging of BODIPY dyes
1

ACCEPTED MANUSCRIPT
A General Modular Approach for the Solubility Tagging of BODIPY
Dyes
Alberto Blázquez-Moraleja, Delia Álvarez-Fernández,† Ruth Prieto Montero,† Inmaculada
García-Moreno, Virginia Martínez-Martínez,* Jorge Bañuelos Prieto, Inés Sáenz-de-Santa-
María, María D. Chiara, and Jose Luis Chiara*
A. Blázquez-Moraleja, D. Álvarez-Fernández, Dr. J. L. Chiara
Instituto de Qu
́mica Orgánica General, IQOG-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
E-mail: jl.chiara@csic.es
R. Prieto Montero, Dr. V. Martínez-Martínez, Dr. J. Bañuelos Prieto
Departamento de Qu mica F sica, Universidad del Pa s Vasco-EHU, Facultad de Ciencias y
Tecnolog a, Apartado 644, 48080 Bilbao, Spain
́
́
́
́
E-mail: virginia.martinez@ehu.eus
Prof. I. García-Moreno
Instituto Qu ́mica-F ́sica “Rocasolano”, IQFR-CSIC, Serrano 119, 28006 Madrid, Spain
Dr. I. Sáenz-de-Santa-María, Dr. M. D. Chiara
Institute of Sanitary Research of Asturias, Institute of Oncology of Asturias (IUOPA),
CIBERONC, Universidad de Oviedo, Hospital Central de Asturias, 33011 Oviedo, Spain
†These authors have contributed equally to this work
Keywords: fluorescent dyes, BODIPYs, aqueous solubility, imaging agents, density functional
calculations
The authors have no competing interests to declare.
Abstract
We describe a general and practical strategy for the direct one-step incorporation of a tunable
solubility module at the boron atom of F-BODIPY dyes. The tethering reaction uses easy-to-
handle reagents, has broad functional group compatibility and proceeds under mild
conditions without requiring any pre-functionalization of the starting F-BODIPY to yield the
solubility-tagged O-BODIPY derivative in 51-86% yield. The module can be introduced at the
end of the synthetic route without perturbing the fluorophore scaffold, thus minimizing
difficulties in product isolation. Its orthogonal geometrical arrangement with respect to the
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plane of the BODIPY chromophore hampers intermolecular aggregation processes that
quench fluorescence, while the covalent attachment to the boron atom has a minimal effect on
the absorption properties. Fully water-soluble and hydrolytically stable BODIPYs were
prepared by incorporating either neutral (tetra- and octaethylene glycol chains) or
zwitterionic (sulfobetaine) hydrophilic tags in the module. The new dyes are valuable live cell
imaging probes that ameliorate the undesired partitioning into lipophilic compartments that
is often observed for standard BODIPYs. This strategy can be readily adapted to the general
and highly practical post-synthetic introduction of new functionalities into F-BODIPY dyes,
including phase-tagging, by appropriately choosing the nature of the chemical tags attached to
the module.
1. Introduction
The expanding development of chromogenic and fluorogenic probes for practical applications
in the study of biosystems critically depends on the facility to post-synthetically introduce
diverse functionalities that improve the analytical and physicochemical properties of the
probe without significantly affecting its photophysical properties. Among commonly used
dyes for biological studies, BODIPYs outstand for their fascinating chemical, electronic, and
photophysical features, comprising large molar absorption coefficients, sharp fluorescence
emissions with high fluorescence quantum yields, excellent photostability, and broad
chemical versatility.[1] However, due to its inherent hydrophobic nature, the standard BODIPY
chromophore has also serious drawbacks for its application in biological research, such as a
very low solubility in aqueous media and a tendency to form aggregates.[2] As a consequence,
BODIPYs often partition into lipophilic compartments of cells resulting in nonspecific staining
of biomembranes and/or cell organelles, which can seriously bias the cellular distribution of
labeled small molecules. Likewise, they also tend to embed into hydrophobic pockets[3] when
conjugated to biomolecules, which may affect the biological function of the bioconjugate.
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Different approaches have been developed along the last decade to improve water solubility
and minimize aggregation of BODIPY dyes, which include the introduction of ionizable or
permanently charged hydrophilic groups, such as anionic carboxylic,[4] phosphonic[4b,4c,5] and
sulfonic acid salts,[4c,6] cationic phosphonium[7] and ammonium groups,[4c,8] and zwitterionic
sulfobetaines,[4,6e,9] or grafting the fluorophore to hydrophilic (bio)/mono-/oligo-/polymers,
such as carbohydrates,[8c,10] peptides,[11] oligonucleotides,[12] and mono-/oligoethylene
glycols.[13] While significantly improving water solubility, the ionic character and/or large size
of the solubilizing groups often impair membrane permeability, which limit the practical
application of the fluorescent tag in live cell and in vivo imaging. In addition, most of these
approaches implement multistep synthetic routes to introduce the desired water-solubilizing
groups, which have to be masked during the synthesis to avoid troublesome purifications
and/or prevent cross reactivity.
Figure 1. General modular approach for the solubility-tagging of F-BODIPYs described in this
work.
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We describe herein a general and practical strategy for the efficient one-step, post-
synthetic introduction of a finely tunable solubility module into F-BODIPY dyes (Figure 1). In
our strategy, the solubility module can be directly introduced at the end of the synthetic route
without perturbing the fluorophore scaffold, thus minimizing difficulties in product isolation.
To this end, we selected a covalent functionalization reaction with a broad functional group
compatibility that proceeds under mild conditions and in high yield without requiring any
pre-functionalization of the chromophore. Such a reaction is the basis of our recently
described one-step synthesis of O-BODIPYs with a rigid B-spiranic 4,4-diacyloxyl or 4-
acyloxyl-4-alkoxyl substitution pattern starting from the corresponding F-BODIPYs.[14] Our
original protocol allows the direct modification of known F-BODIPY dyes at the boron atom
via an efficient F-to-O exchange reaction that uses simple reagents and conditions, providing
an easy access to diversely functionalized mono-[14a] and bichromophoric[14b] O-BODIPYs. In
addition, the conformational rigidity of the resulting B-spiranic system boosts fluorescence
quantum yield and photochemical stability, while the orthogonal disposition of the B-chelated
di-O-substituent with respect to the mean plane of the boradiazaindacene framework hinders
intermolecular π-stacking aggregation of the chromophores, which impede the formation of
H-dimers that strongly quench fluorescence. Moreover, substitution at boron usually has a
minimal effect on the absorbance and emission properties of the BODIPY chromophore.
Starting from known F-BODIPYs 1-3, we have validated our strategy by designing a rigid
water-solubilizing module based on 2,4,6-trihydroxybenzoic acid (phloroglucinol carboxylic
acid, 4) as the core, incorporating two identical hydrophilic side chains of different nature,
neutral or zwitterionic, to promote water solubility, thus allowing the use of our dyes in
aqueous biological environments (Figure 1).
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2. Results and Discussion
2.1. Synthetic approach to solubility-tagged BODIPYs. The synthesis route to the different
solubility-tagged modules and the final tagged water-soluble BODIPYs is shown in Scheme 1.
First, the salicylic acid portion of 4, which will be covalently attached to boron in the final dye,
was protected as an acetonide ester 5 following a literature procedure.[15] The hydrophilic
tags were then introduced in one step using a Mitsunobu protocol[15,16] to afford the protected
modules 6a-c. As hydrophilic tags, we have chosen two neutral oligoethylene glycol chains of
different lengths, based on commercially available tetra- and octaethylen glycol monomethyl
ether (R1O = a and b, respectively), and a zwitterionic (sulfobetaine) chain (R1O = c, Figure 1)
that was readily prepared from 2-[2-(dimethylamino)ethoxy]etanol and propane sultone
using a modification of a described procedure.[17] Removal of the acetonide protecting group
under carefully controlled hydrolytic conditions,[18] afforded the final hydrophilic modules
7a-c ready for tethering to F-BODIPYs. As commented above, these modules were then
directly incorporated into the F-BODIPYs 1-3 included in this work, using our recently
described one-step protocol.[14] Thus, a stoichiometric mixture of F-BODIPY and the
corresponding module 7a-c in anhydrous MeCN was heated under microwave irradiation
(50-120 °C, 0.75-2 h) in the presence of an excess of TMSCl (20-50 equiv) to afford the
targeted water-soluble O-BODIPYs 1a-b, 2a-c, and 3a-c in moderate to good yield (51-86%),
after solvent removal and chromatographic purification (Scheme 1). This F-to-O exchange
method uses an easy-to-handle and volatile reagent, avoids aqueous work-up (the TMSCl
reagent and the corresponding TMSF only by-product are both volatile) and is experimentally
simpler, more efficient, and yields cleaner reaction crudes than all previously reported
methodologies to prepare O-BODIPYs.[13e,19]
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Scheme 1. Synthesis of the water-solubilizing modules and the final water-soluble BODIPYs
(see Figure 1 for the chemical structures of the R substituents).
2.2. Photophysical characterization.
A comprehensive computationally-aided photophysical study was performed on the newly
synthesized O-BODIPYs. At a first sight, the solubility-tagged module linked to the boron atom
of the BODIPY skeleton should not significantly affect the photophysics of the new dyes.[14a,20]
Indeed, in related O-BODIPYs the presence of different spiranic rings centered at the boron
bridge neither alters the absorption profile nor the fluorescence signatures, following thereby
a photophysical behavior similar to their respective parent F-BODIPY precursors.[14a,,19d,21]
Strikingly, upon addition of the hydrophilic chains to the phenyl group of the solubility
module some photophysical properties become strongly dependent on both the substitution
pattern of the chromophoric core and the solvent polarity. The hydrophilic chains did not
alter the absorption properties (band-shape, absorption coefficient and maximum
wavelength) nor their typical solvent dependence, hypsochromically shifting the maximum
wavelength and reducing the molar extinction coefficient on increasing solvent
polarity.[14a,19d,21] However, they induced a dramatic effect on the fluorescent emission of the
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new dyes (Tables 1 and S1). In particular, dyes 1a-b exhibited low fluorescence quantum
yields and short lifetimes, which are far from the typical values of similar 8-mesityl
substituted F-BODIPYs (Table 1 and S1).[22]
Table 1. Photophysical properties of dyes 1b, 2b and 3b in different solvents.a) The
photophysical data for the other dyes are collected in Tables S1-S3.
τf)
(ns)
b)
c)
d)
λab
εmax
λfl
e)
Dye
1b
Solvent
φ
flu
(nm)
(104
M
–1 cm–1)
(nm)
CHCl3
EtOH
H2O
507.0
5.8
5.1
4.0
521.0
0.06
0.02
0.49
0.11
505.0
503.0
507.0
504.0
502.0
527.0
523.0
520.0
514.5
514.0
515.5
514.5
511.5
547.0
545.5
540.0
<0.01
<0.15
2b
3b
CHCl3
EtOH
H2O
7.8
6.9
4.2
6.2
4.5
4.9
0.86
0.79
5.82
6.60
6.40
6.85
7.28
7.77
0.78
CHCl3
EtOH
H2O
0.73g
0.72g
0.71g
b)
c)
a)Dye concentration: 2×10–6
M. Absorption peak wavelength. Molar extinction coefficient at
peak. d)Fluorescence peak wavelength, upon excitation at 490 nm. e)Using quantum yield of
f)
g
PM546 (
φ
fl = 0.91) in cyclohexane as reference. Fluorescence lifetime. Using quantum yield
of PM567 (
φ
fl
= 0.84) in ethanol as reference. The fluorescence data are recorded in right
angle using a 1 cm optical path cell.
Since the low emission efficiency of dyes 1a and 1b could not be attributed to the
molecular flexibility of the 8-aryl substituent,[22] we hypothesized that its fluorescent
behavior may be related to the high electron density of the substituent grafted at the boron
center (a phenyl group decorated with three electron-donor oxygen atoms). Dramatic
fluorescence quenching in O-BODIPYs has been already reported upon introduction of a
spiranic ring at the boron bridge (e.g. catechol or binol).[23] Such derivatives are weakly
emissive due to the activation of intramolecular charge transfer (ICT) processes, which could
7

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eventually evolve into a photoinduced electron transfer (PET) as the main non-radiative
deactivation channel.[23] Trying to confirm this hypothesis, we carried out DFT atomistic
simulations (B3LYP/6-311+G*) of dye 1a and its methylated analog 2a as representative
compounds (Figure 2). The HOMO–1 of the non-methylated BODIPY 1a, almost entirely
located on the pendant aromatic moiety grafted at the boron atom, is energetically very close
to the HOMO (just 0.08 eV below), which is completely located on the dipyrrin system. Such
energetic distribution of molecular orbitals envisaged the ability of the electron-rich salicylic
system to effectively induce an intramolecular charge transfer (ICT) process and even enable
a reductive photoinduced electron transfer (PET) upon excitation. Thus, upon the
photoinduced promotion of an electron from the HOMO to the LUMO, an electron transfer
from the low-lying HOMO–1 to the semi-vacant HOMO is thermodynamically feasible. Such a
reductive PET from the spiranic group grafted at boron to the BODIPY core avoids radiative
deactivation from the LUMO back to the HOMO, thus explaining the almost negligible
fluorescence emission of 1a. The presence of alkyl groups on the chromophoric core of 2a,
drastically changes the energetic arrangement of the molecular orbitals. Thus, with respect to
dye 1a, the inductive electron-donor ability of the methyl groups in 2a increases the energy of
the HOMO and LUMO orbitals (by ca. 0.56 eV and ca. 0.35 eV, respectively) without
energetically altering the HOMO–1. Consequently, the energy gap between the frontier
orbitals HOMO–1 and HOMO (0.58 eV) becomes 7-fold higher than in the non-methylated
counterpart 1a, thus hampering an effective PET process and thereby allowing the
fluorescence emission of the BODIPY. Therefore, tetramethylation of the BODIPY scaffold
reduces the electron-acceptor character of the chromophoric core making thermodynamically
unfeasible any ICT process induced by the functionalization at its boron bridge. Thus, the
bright fluorescence emission and long excited-state lifetimes of typical BODIPYs are
recovered in dyes 2a-c (Tables 1 and S2).[14a,19d,20-22] In the same manner, the electron-donor
character of the ethyl groups additionally grafted at the C-2 and C-6 positions of BODIPYs 3a-c
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further reduces the probability of an ICT process, allowing these dyes to emit highly efficient
fluorescence with elongated lifetimes regardless of the length and nature of the hydrophilic
chains and the solvent polarity (Tables 1 and S3).
Figure 2. Calculated (B3LYP/6-311+G*) energetic arrangement of the frontier molecular
orbitals of the non-methylated BODIPY 1a and its tetramethylated analog 2a.
In addition, the alkyl substituents on the BODIPY core may also play a major role in
controlling intermolecular interactions. The capability of the new dyes to avoid non-
fluorescent aggregation in water would impel their application in biological settings, since
fluorescent probes are typically used in concentrations higher than 2 µM. To understand the
intermolecular interactions of dyes 2a-c and 3a-c in aqueous media, the photophysical
studies were also performed at increasing concentrations of dye in water (see Experimental
Section for details). The fluorescence quantum yields of 2a-c and 3a-c (approximate values at
the highest concentrations) were not significantly affected by augmenting dye concentration
from 2 × 10–6
M M, which emphasized the absence of aggregates and the enhanced
to 10–4
water solubility of the new dyes (Tables 2, S4 and S5). A further increase in concentration led
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to the gradual quenching of the emission efficiency, which could be ascribed, at least to some
extent, to the strengthening of reabsorption/reemission processes (Table S4 and S5).[24] In
addition, growing concentrations markedly changed the spectral profiles depending on the
substitution pattern of the chromophoric backbone. In the case of 2a-c, an increase of dye
concentration in water produced a gradual broadening and a slight red-shift of the absorption
spectra, without entailing neither the growth of vibronic shoulders nor the appearance of new
bands (Figure 3). Likewise, unusual features emerged in the emission spectra, with the
intensity of the long-wavelength shoulder growing with dye concentration (Figure 3) until
reaching or even exceeding the intensity of the main fluorescence band (Figure S3).
Interestingly, the fluorescence lifetime was never significantly shortened in highly
concentrated solutions with respect to that recorded in diluted solutions (Tables 2 and S4).
This spectral behavior is fingerprint of short-range intermolecular interactions, likely
excimer-like species and/or J-type aggregates.[14a]
Table 2. Dependence of the fluorescence quantum yield and lifetime of new derivatives 2ca)
and 3cb) on dye concentration in water. The data for the other dyes are collected in Tables S4-
S5.
Dye 2c
φd)
Dye 3c
φd)
[c]x104
(M)
0.02
0.05
0.1
0.2
0.3
0.7
0.9
1.6
2.3
3.5
7.0
φc)
τ
(ns)c)
τ
(ns)d)
φc)
τ
(ns)c)
τ
(ns)d)
0.86
0.85
0.86
0.85
0.83
0.87
-
5.99
6.09
6.19
6.36
-
-
-
-
-
-
-
-
-
-
0.78
0.78
0.78
0.76
0.78
-
0.68
0.35
0.22
0.06
0.01
-
-
-
-
-
-
7.25
7.27
7.36
7.49
7.70
-
-
-
-
-
-
-
-
0.77
0.48
0.34
0.24
0.09
7.88
8.22
8.24
-
0.45 0.58
6.76
6.25
-
6.47
-
7.48
7.47
7.21
-
-
-
-
-
-
0.19 -
-
-
-
a) Upon excitation at 490 nm and using PM546 (
excitation at 490 nm and using PM567 (
front-face configuration using 1 and 0.1 mm optical path cuvette, respectively.
φ
fl = 0.91) in cyclohexane as reference. b) Upon
φ
fl =0.84) in ethanol as reference. c,d) Evaluated under
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Figure 3. Height-normalized absorption (A) and emission (B) spectra of compound 2c in
water at different concentrations: a) 2.0×10–6
M M M
(1cm); b) 1.0×10–5 (1mm); c) 1.7×10–4
(0.1mm); d) 3.4×10–4
M (0.1mm). The corresponding spectra for 2a-b dyes are collected in
Figures S1 and S2.
In contrast to 2a-c, the absorption spectra of 3a-c are strongly dependent on dye
concentration (>10–4
M), with the vibronic shoulder placed at 490 nm growing with
concentration until reaching the intensity of the main absorption band centered at 520 nm
(Figure 4). Conversely, the emission spectral profile of dyes 3a-c remained roughly
unchanged except for a decrease of the fluorescent intensity and a slight red-shift of the main
fluorescence band. This photophysical behavior (Tables 2 and S5) is indicative of not emissive
H-type intermolecular interactions, which efficiently quench the fluorescence emission. The
more planar structure of the boradiazaindacene unit in dyes 3a-c allows an effective
intermolecular π-stacking arrangement of the chromophores at high concentrations favored
by the low steric hindrance of the 8-methyl group as compared to the orthogonally oriented
8-mesityl group in dyes 2a-c. Thus, compounds 2a-c displayed higher fluorescence quantum
yields and shorter lifetimes than dyes 3a-c regardless of dye concentration and solvent
polarity.
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Figure 4. Height-normalized absorption (A) and emission (B) spectra for compound 3c in
water at different concentrations: a) 2.2×10–5
M
; b) 1.6×10–4
M
, recorded in 1-mm optical path
cuvette; c) 2.3×10–4
M
; d) 3.9×10–4
M
; e) 6.8×10–4 ; f) 7.4×10–4
M
M, recorded in 0.1 mm optical
path cuvette. The corresponding spectra for 3a-b dyes are collected in Figures S3 and S4.
2.3. Solubility, chemical stability and lipophilicity.
For their intended use in live cell imaging, we have studied the solubility, stability in water,
and lipophilicity of the tagged dyes. All the new BODIPYs were completely soluble in water at
10–4
studies. As a representative example, compound 3b was very soluble in water with a
determined maximum solubility of 10.0 ± 0.1 m at room temperature. As expected, the di-
M, which is well above the maximum concentration typically required for cell microscopy
M
charged sulfobetaine derivatives 2c and 3c were even more water-soluble, but maximum
solubility values could not be reached in this case due to lack of sufficient material. In
comparison, compounds 1a, 2a and 3a, with the smallest neutral solubility tag, required co-
addition of DMSO to fully dissolve in water in the millimolar range (see below). Thus, our
modular one-step approach allows to fine tune the solubility properties of the attached
BODIPY dye by a simple and efficient postsynthetic transformation.
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Since the new tagged dyes contain two potentially labile B—O bonds connecting the
fluorophore to the solubility module, we also monitored their chemical stability in aqueous
media. Thus, we studied the evolution of BODIPYs 1a, 2a and 3a, a set of dyes sharing the
same solubility module, under neutral aqueous conditions (10 mM solution in 1:1 D2O/DMSO-
1
d6, at room temperature) using H NMR. The compounds were completely stable under the
1
tested conditions with no trace of hydrolysis products being observed in the H NMR spectra
of the aged solutions even after 30 days at room temperature. For their intended application
in cell microscopy, we have also studied the stability under simulated physiological conditions
(cell culture medium with 10% fetal bovine serum, pH 7.4) and in phosphate buffered saline
at pH 6.0 and 8.0, at room temperature, using again BODIPY 3b as representative example. No
changes in absorbance nor lineshape were observed in the UV-visible spectra of the
corresponding solutions after monitoring for 7 days under these conditions (see Figures S5-
S7, Supplementary Material), attesting to the very high chemical stability of these BODIPYs in
the most relevant aqueous media used in biological studies. The high stability towards
hydrolysis observed for these new BODIPYs probably results from the bidentate covalent
binding of the salicylic module to the boron atom of the dye.
To better understand transmembrane transport and distribution of the new dyes
between aqueous and hydrophobic subcellular compartments, we also assessed their
lipophilicity (expressed by log P). Due to the limiting amount of sample, we employed the
reversed-phase high-performance liquid chromatography (RP-HPLC) method with a C18
analytical column (Table 3 and Figure S8) to determine log P.[4d,25] The log P values measured
for the new series of BODIPYs (log P = –1.51 to 4.22) fit the range of biological applications
defined by Lipinski (log P ≤ 5)[26] and were also approximately within the same range
reported for other water-soluble BODIPY dyes (log P = –0.3 to 4.5).[4d,13e,27] For the same
starting BODIPY, lipophilicity decreased with increasing aqueous solubility in the order: a
(tetraethylene glycol chains) > b (octaethylene glycol chains) > c (sulfobetaine chains), as
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generally expected. For the same solubility tag, lipophilicity decreased in the order: 2 > 3 > 1
for each a-c series of compounds, which is the same order theoretically predicted for the
parent F-BODIPYs (CLogP = 7.56, 7.00, and 5.17, respectively, calculated with ChemBioDraw).
Table 3. Retention factors and partition coefficients of selected reference compounds used
for calibration and the new BODIPY dyes.a)
Compound
Retention factorb,c k/log k
10.16/1.01
3.09/0.49
log P
5.4
2,6-di-tert-butyl-4-methylphenol
Diphenylmethane
4.1
1,2-Dicholorobenzene
2.09/0.32
3.4
Bromobenzene
1.66/0.22
3.0
Toluene
1.48/0.17
2.7
4-Chlorophenol
0.45/–0.35
0.26/–0.58
0.14/–0.87
1.88/0.27
2.4
Phenol
1.5
Pyrocatechol
0.9
1a
1b
2a
2b
2c
3a
3b
3.44d)
2.90d)
4.22d)
3.66d)
–1.51d)
3.58d)
1.11/0.04
3.99/0.60
2.31/0.36
0.02/–1.81
2.13/0.33
1.19/0.07
2.97d)
e)
e)
3c
a)
RP-HPLC analyses were performed at 25 °C using 0.1% aq. trifluoroacetic acid/CH3CN
(65:35) as mobile phase. b) Calculated with the equation k = (tR – t0)/t0, where t0 is the dead
time (determined by injection of an unretained organic compound: uracil, t0 = 0.58 min).
c)
d)
Average values of three repeated injections.
Calculated with the calibration (linear
regression) equation: log P = (log k + 1.175)/0.42. e) An appropriate retention factor could not
14

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be obtained for 3c under the selected conditions due to its very fast elution from the column.
Log P for this compound is then lower than –1.5, the value estimated for the less retained
compound 2c.
2.4. Live cell imaging studies.
To assess if this modular strategy could not only successfully modify the solubility properties
of the tagged BODIPYs, but also ameliorate the lipophilicity and aggregation pitfalls that
common BODIPYs usually present in biological applications, we have studied the behavior of
the new dyes in live cell microscopy. To this end, live SCC38 cells (derived from human larynx
squamous cell carcinoma) were incubated with aqueous solutions of 1a-b, 2a-c, and 3a-c at
different concentrations (50, 100 and 500 nM). The observed staining behavior depended on
both, the structure of the chromophoric subunit and the tethered solubility module. For the
same solubility module, fluorescence brightness decreased in the order 2a-b > 3a-b > 1a-b
(Figure 5), which, not surprisingly, is the same order observed in our prior photophysical
study in water solution. In fact, fluorescent staining with 1a-b was too weak to be of practical
use even at the highest concentration assayed. Regarding the solubility module, no staining
could be observed for BODIPYs tethered with double-charged sulfobetaines (2c and 3c),
which were not able to cross the cell membrane even after prolonged incubation, as recently
observed for other dyes containing similar sulfobetaine substituents.[28] For the neutral
BODIPYs, the longer the oligoethylene chain, the less cell-penetrating the BODIPY dye was.
Thus, BODIPYs with the long octaethylene glycol chains (2b and 3b) showed a weaker cell
fluorescence signal, which could only be clearly observed for the larger and most lipophilic
BODIPY 2b at the highest concentration assayed (500 nM). In all cases, cell staining was rather
diffuse, but some subcellular perinuclear reticulate staining pattern could still be observed
that is consistent with partial nonspecific binding to the ER and Golgi for this series of dyes.
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Figure 5. Representative fluorescence images and relative mean fluorescence intensities of
live SCC38 cells incubated with the indicated concentrations of the new solubility-tagged O-
BODIPYs for 30 min at 37 °C before washing and microscopic analysis. To compare
fluorescence intensities for the different dyes and concentrations, all images were taken with
the same exposure time (200 ms) on the same day. No staining could be observed for
BODIPYs 2c and 3c, even at the highest concentration assayed (500 nM). Scale bars: 10 µm.
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3. Conclusions
In this study, we have developed a modular one-pot synthetic strategy for the direct post-
functionalization of F-BODIPYs with a structural subunit designed to fine-tune their solubility
properties. This solubility module is based on a 2,4,6-trihydroxybenzoic acid scaffold onto
which two solubility-tags are readily incorporated in three simple synthetic steps with good
overall yield. The tethering reaction to the starting F-BODIPY dye uses easy-to-handle
reagents, has broad functional group compatibility, and proceeds under mild conditions and
in high yield without requiring any pre-functionalization of the chromophore, thus allowing
the efficient incorporation of the solubility module at the end of the synthetic route without
perturbing the chromophore scaffold. The orthogonal geometrical arrangement of this
module with respect to the plane of the BODIPY chromophore hampers intermolecular
aggregation processes that quench fluorescence. The covalent attachment of the module to
the boron atom has a minimal effect on the absorption properties, while its effect on the
fluorescence emission properties depends on the degree of alkylation of the chromophoric
system. Thus, in the case of the non-alkylated chromophores derived from parent F-BODIPY 1,
low fluorescence quantum yields are observed due to deactivation by a thermodynamically
facile ICT process, as predicted by DFT atomistic simulations. In comparison, alkylation of the
BODIPY scaffold reduces its electron-acceptor character making any ICT process
thermodynamically unfeasible and yielding highly efficient fluorescence, as observed for the
dyes derived from F-BODIPYs 2 and 3.
Fully water-soluble and hydrolytically stable BODIPYs have been prepared by
incorporating hydrophilic chemical tags in the solubility module. The new water-soluble O-
BODIPYs containing either neutral (tetra- and octa-ethylene glycol chains) or zwitterionic
(sulfobetaine) tags are highly stable under the most common aqueous conditions used in
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biological settings (pH 6-8). Live cell imaging studies of the new dyes give very diffuse cell
staining with only minor subcellular perinuclear reticulate staining, attesting to their reduced
lipophilicity as compared with typical BODIPY dyes. The observed cell membrane
permeability depends on the nature and size of the chemical tags present in the solubility
module, decreasing in the order: tetraethylene glycol > octaethylene glycol >> sulfobetaine.
The new strategy can be readily adapted to the general and highly practical post-synthetic
introduction of new functionalities into F-BODIPYs, including phase-tagging, by appropriately
choosing the nature of the chemical tags attached to the module.
4. Experimental Section
4.1. General methods.
Proton and carbon-13 nuclear magnetic resonance (1H NMR or 13C NMR) spectra were
recorded on a Bruker Avance III-400 (400 and 100 MHz, respectively) or a Varian System 500
(500 and 125 MHz, respectively) spectrometers. Chemical shifts are expressed in parts per
million (δ scale) downfield from tetramethylsilane and are referenced to residual peaks of the
deuterated NMR solvent used. Data are presented as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, m = multiplet and/or multiple resonances, b = broad),
integration, coupling constants in hertz (Hz), and assignment. Proton and carbon-13
assignments are based on gCOSY, gHSQC, and gHMBC correlation experiments. Thin layer
chromatography (TLC) was performed with Merck Silica Gel 60 F254 plates and Merck Silica
Gel 60 RP-18 F254S plates. Chromatograms were visualized using UV light (254 nm or 365
nm). Column chromatography was performed on a 971-FP Flash Purification System from
Agilent Tecnologies using SF Si35 or C18 cartridges. High-resolution mass spectra (HRMS)
were recorded on an Agilent 6520 Q-TOF instrument with an ESI source. Anhydrous solvents
were prepared according to standard methods by distillation over drying agents or via elution
through a PureSolv™ column drying system from Innovative Technology, Inc. All other
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solvents were of HPLC grade and were used as provided. All reactions were carried out with
magnetic stirring and, if air or moisture sensitive, in oven-dried glassware under argon.
Microwave irradiation experiments were performed with a single-mode Anton Parr
Monowave 300 reactor, using standard Pyrex tubes (10 mL capacity) sealed with a PTFE-
lined rubber septum.
4.2. Synthesis of the solubility-tagged BODIPYs
BODIPY 1a. A solution of F-BODIPY 1 (18.70 mg, 0.061 mmol), 7a (33.30 mg, 0.061
mmol), and trimethylsilyl chloride (154 µL, 1.21 mmol) in dry acetonitrile (2.50 mL) was
heated under microwave irradiation at 120 °C for 90 minutes. The solvent and excess volatile
reagents were removed at reduced pressure and the resulting residue was purified by flash
chromatography (CH2Cl2/MeOH 100:0 → 90:10) to afford 1a (29 mg, 51%) as a red solid.
1H NMR (CDCl3, 400 MHz): δ = 7.69 (2H, s, H3 and H5), 6.95 (2H, s, H3´ and H5´), 6.68 (2H, d, J
= 4.2 Hz, H1 and H7), 6.40 (2H, dd, J = 4.2, 1.9 Hz, H2 and H6), 6.16 (1H, d, J = 2.3 Hz, H6´´),
6.08 (1H, d, J = 2.3 Hz, H4´´), 4.22 (2H, t, J = 5.2 Hz, H8´´), 4.11 (2H, t, J = 4.7 Hz, H17´´), 3.99
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(2H, t, J = 5.2 Hz, H9´´), 3.87 (2H, t, J = 4.7 Hz, H18´´), 3.84 (2H, br t, H10´´), 3.72-3.62 (18H, m,
H11´´-H14´´ and H19´´-H23´´), 3.55-3.52 (4H, m, H24´´ and H15´´), 3.36 (3H, s, H16´´/H25´´),
3.36 (3H, s, H25´´/H16´´), 2.36 (3H, s, CH34´), 2.14 (3H, s, CH32´/CH36´), 2.11 (3H, s,
CH36´/CH32´). 13C NMR (CDCl3, 100 MHz): δ = 164.6 (C3´´), 162.3 (C7´´), 162.2 (C5´´), 160.8
(C1´´), 147.9 (C8), 144.7 (C3 and C5), 139.0 (C4´), 136.7 (C2´/C6´), 136.3 (C6´/C2´), 135.3 (C7a
and C8a), 130.6 (C1 and C7), 129.7 (C1´), 128.4 (C3´/C5´), 128.2 (C5´/C3´), 118.8 (C2 and C6),
99.2 (C2´´), 95.6 (C4´´), 94.3 (C6´´), 72.1 (C15´´ and C24´´), 71.2 (C10´´), 71.0, 70.8, 70.8, 70.8,
70.8, 70.7, 70.7, 70.6, 70.6 (from 71.0 to 70.6: C11´´-C14´´ and C19´´-C23´´), 69.6 (C18´´), 69.4
(C9´´), 69.2 (C8´´), 67.7 (C17´´), 59.2 (C16´´/C25´´), 59.2 (C25´´/C16´´), 21.3 (CH34´), 20.3
(CH32´/CH36´), 20.2 (CH36´/CH32´). HRMS (API-ES+) calcd. for C43H58BN2O13 (M+H)+
821.4034; found 821.4055; Calcd. for C43H57BN2NaO13 (M+Na)+ 843.3853; found 843.3879.
BODIPY 2a. A solution of F-BODIPY 2 (21.20 mg, 0.058 mmol), 7a (26.20 mg, 0.048
mmol), and trimethylsilyl chloride (302 µL, 2.38 mmol) in dry acetonitrile (2.50 mL) was
heated under microwave irradiation at 120 °C for 1 h. The solvent and excess volatile reagents
were removed at reduced pressure and the resulting residue was purified by flash
chromatography (CH2Cl2/MeOH 100:0 → 90:10) to afford 2a (35.90 mg, 86%) as a red solid.
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1H NMR (CDCl3, 400 MHz): δ = 6.95 (2H, s, H3´ and H5´), 6.10 (1H, d, J = 2.3 Hz, H6´´), 6.05 (1H,
d, J = 2.3 Hz, H4´´), 5.92 (2H, s, H2 and H6), 4.19 (2H, t, J = 5.2 Hz, H8´´), 4.10 (2H, t, J = 4.9 Hz,
H17´´), 3.98 (2H, t, J = 5.2 Hz, H9´´), 3.87-3.83 (4H, m, H10´´ and H18´´), 3.73-3.63 (18H, m,
H11´´-H14´´ and H19´´-H23´´), 3.55-3.53 (4H, m, H15´´ and H24´´), 3.37 (3H, s, H16´´/H25´´),
3.36 (3H, s, H25´´/H16´´), 2.33 (3H, s, CH34´), 2.19 (6H, s, CH33 and CH35), 2.14 (3H, s,
CH32´/CH36´), 2.11 (3H, s, CH36´/CH32´), 1.37 (6H, s, CH31 and CH37). 13C NMR (CDCl3, 100
MHz): δ = 164.7 (C7´´), 163.5 (C5´´), 162.2 (C1´´), 161.3 (C3´´), 155.8 (C3 and C5), 142.8 (C7a
and C8a), 142.0 (C8), 138.8 (C4´), 135.3 (C2´/C6´), 134.9 (C2´/C6´), 131.3 (C1´), 131.2 (C1 and
C7), 129.3 (C3´/C5´), 129.1 (C5´/C3´), 121.9 (C2 and C6), 99.7 (C2´´), 94.6 (C4´´), 93.7 (C6´´),
72.1 (C15´´/C24´´), 72.1 (C24´´/C15´´), 71.2 (C10´´), 71.0, 70.8, 70.8, 70.8, 70.7, 70.7, 70.6, 70.6
(from 71.0 to 70.6: C11´´-C14´´ and C19´´-C23´´), 69.6 (C18´´), 69.3 (C9´´), 69.1 (C8´´), 67.6
(C17´´), 59.2 (C16´´/C25´´), 59.2 (C25´´/C16´´), 21.3 (CH34´), 19.9 (CH32´/CH36´), 19.7 (CH36´/
CH32´), 15.5 (CH33 and CH35), 13.7 (CH31 and CH37). HRMS (API-ES+) calcd. for C47H66BN2O13
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(M+H)+ 877.4660; found 877.4643; Calcd. for C47H65BN2NaO13 (M+Na)+ 899.4480; found
899.4494.
BODIPY 3a. A solution of F-BODIPY 3 (17.84 mg, 0.056 mmol), 7a (32.73 mg, 0.059
mmol), and trimethylsilyl chloride (151 µL, 1.19 mmol) in dry acetonitrile (2.50 mL) was
heated under microwave irradiation at 120 °C for 90 minutes. The solvent and excess volatile
reagents were removed at reduced pressure and the resulting residue was purified by flash
chromatography (CH2Cl2/MeOH 100:0 → 90:10) to afford 3a (31.80 mg, 65%) as an orange
solid.
1H NMR (CDCl3, 400 MHz): δ = 6.09 (1H, d, J = 2.2 Hz, H6′), 6.00 (1H, d, J = 2.2 Hz, H4′), 4.18
(2H, t, J = 5.3 Hz, H8´), 4.08 (2H, t J = 4.7 Hz, H17´), 3.98 (2H, t, J = 5.3 Hz, H9´), 3.86-3.82 (4H,
m, H10´ and H18´), 3.71-3.62 (18H, m, H11´´-H14´´ and H19´´-H23´´), 3.55-3.53 (4H, m, H15´
and H24´), 3.37 (3H, s, H16´/H25´), 3.36 (3H, s, H25´/ H16´), 2.62 (3H, s, CH38), 2.34 (4H, q, J =
7.5 Hz CH3CH22 and CH3CH26), 2.33 (3H, s, CH31/CH37), 2.32 (3H, s, CH37/CH31), 2.13 (6H, s,
CH33 and CH35), 0.98 (6H, t, J = 7.5 Hz, CH3CH22 and CH3CH26). 13C NMR (CDCl3, 100 MHz): δ =
164.6 (C5´), 163.5 (C3´), 162.3 (C7´), 161.1 (C1´), 152.5 (C3 and C5), 140.0 (C8), 137.0 (C7a
and C8a), 133.0 (C1 and C7), 132.4 (C2 and C6), 100.0 (C2´), 94.7 (C4´), 93.6 (C6´), 72.1 (C15´
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and C24´), 71.2 (C10´), 71.0, 70.9, 70.8, 70.8, 70.7, 70.7 (from 71.0 to 70.7: C11´´-C14´´ and
C19´´-C23´´), 69.6 (C18´), 69.4 (C9´), 69.1 (C8´), 67.6 (C17´), 59.2 (C16´/C25´), 59.2
(C25´/C16´), 17.4 (CH38), 17.3 (CH3CH22 and CH3CH26), 15.1 (CH3CH22 and CH3CH26), 14.7
(CH31 and CH37), 13.01 (CH33 and CH35). HRMS (API-ES+) calcd. for C43H66BN2O13 (M+H)+
829.4660; found 829.4679; Calcd. for C43H65BN2NaO13 (M+Na)+ 851.4479; found 851.4494.
BODIPY 1b. A solution of F-BODIPY 1 (13.98 mg, 0.045 mmol), 7b (37.0 mg, 0.045
mmol), and trimethylsilyl chloride (132 µL, 1.02 mmol) in dry acetonitrile (2.50 mL) was
heated under microwave irradiation at 120 °C for 90 minutes. The solvent and excess volatile
reagents were removed at reduced pressure and the resulting residue was purified by flash
chromatography (CH2Cl2/MeOH 100:0 → 90:10) to afford 1b (35 mg, 72%) as a red solid.
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1H NMR (CDCl3, 400 MHz): δ = 7.69 (2H, s, H3 and H5), 6.96 (2H, s, H3´and H5´), 6.69 (2H, br d,
H1 and H7), 6.41 (2H, dd, J = 4.2, 1.9 Hz, H2 and H6), 6.16 (1H, d, J = 2.2 Hz, H6´´), 6.09 (1H, d, J
= 2.2 Hz, H4´´), 4.21 (2H, t, J = 5.1 Hz, H8´´), 4.11 (2H, t, J = 4.7 Hz, H25´´), 3.99 (2H, t, J = 5.1 Hz,
H9´´), 3.90-3.82 (4H, m, H10´´ and H26´´), 3.73-3.62 (50H, m, H11´´-H22´´ and H27´´-H39´´),
3.58-3.53 (4H, m, H23´´ and H40´´) 3.37 (6H, s, H24´´ and H41´´), 2.36 (3H, s, CH34´), 2.14 (3H,
s, CH32´/CH36´), 2.11(3H, s, CH36´/CH32´). 13C NMR (CDCl3, 100 MHz): δ = 164.6 (C5´´), 162.3
(C7´´), 162.2 (C3´´), 160.7 (C1´´), 147.9 (C8), 144.7 (C3 and C5), 139.0 (C4´), 136.7 (C2´/C6´),
136.3 (C6´/C2´), 135.3 (C7a and C8a), 130.6 (C1 and C7), 129.7 (C1´), 128.4 (C3´/C5´), 128.3
(C5´/C3´), 118.8 (C2 and C6), 99.2 (C2´´), 95.6 (C4´´), 94.3 (C6´´), 72.1 (C23´´ and C40´´), 71.3
24

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(C10´´), 71.0, 70.9, 70.8, 70.8, 70.7, 70.7, 70.6 (from 71.9 to 70.6: C11´´-C22´´ and C27´´-C39´´),
69.6 (C26´´), 69.4 (C9´´), 69.2 (C8´´), 67.7 (C25´´), 59.2 (C24´´ and C41´´), 21.3 (CH34´), 20.3
(CH32´/CH36´), 20.2 (CH36´/CH32´). HRMS (API-ES+) calcd. for C59H93BN3O21 (M+NH4)+
1190.6399; found 1190.6362.
BODIPY 2b. A solution of F-BODIPY 2 (24.54 mg, 0.067 mmol), 7b (55.0 mg, 0.061
mmol), and trimethylsilyl chloride (197 µL, 1.52 mmol) in dry acetonitrile (2.50 mL) was
heated under microwave irradiation at 120 °C for 45 minutes. The solvent and excess volatile
reagents were removed at reduced pressure and the resulting residue was purified by flash
chromatography (CH2Cl2/MeOH 100:0 → 70:30) to afford 2b (35.90 mg, 80%) as a red solid.
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1H NMR (CDCl3, 400 MHz): δ = 6.95 (2H, s, H3´ and H5´), 6.09 (1H, d, J = 2.2 Hz, H6´´), 6.04 (1H,
d, J = 2.2 Hz, H4´´), 5.92 (2H, s, H2 and H6), 4.18 (2H, t, J = 5.2 Hz, H8´´), 4.09 (2H, t, J = 4.7 Hz,
H25´´), 3.98 (2H, t, J = 5.2 Hz, H9´´), 3.87-3.82 (4H, m, H26´´ and H10´´), 3.72-3.70 (2H, m,
H27´´), 3.68-3.62 (48H, m, H11´´-H22´´ and H28´´-H39´´), 3.55-3.52 (4H, m, H23´´ and H40´´),
3.36 (6H, s, H24´´ and H41´´), 2.33 (3H, s, CH34´), 2.19 (6H, s, CH33 and CH35), 2.14 (3H, s,
CH32´/CH36´), 2.10 (3H, s, CH36´/CH32´), 1.37 (6H, s, CH31 and CH37). 13C NMR (CDCl3, 100
MHz): δ = 164.7 (C5´´), 163.5 (C4´´), 162.2 (C7´´), 161.2 (C1´), 155.8 (C4 and C5), 142.8 (C7a
and C8a), 142.0 (C8), 138.8 (C4´), 135.3 (C2´), 134.9 (C6´), 131.2 (C1/C7), 131.2 (C7/C1),
129.2 (C3´/C5´), 129.1 (C5´/C3´), 121.8 (C2 and C6), 99.7 (C2´´), 94.5 (C4´´), 93.7 (C6´´), 72.1
(C23´´ and C40´´), 71.2 (C10´´), 71.0 (C27´´), 70.8, 70.8, 70.7, 70.7, 70.6, 70.6 (from 70.8 to
70.6: C11´´-C22´´ and C28´´-C39´´) 69.6 (C26´´), 69.3 (C9´´), 69.2 (C8´´), 67.6 (C25´´), 59.2
(C24´´ and C41´´), 21.3 (CH34´), 19.8 (CH32´/CH36´), 19.7 (CH36´/CH32´), 15.5 (CH33 and
CH35), 13.6 (CH31 and CH37). HRMS (API-ES+) calcd. for C63H98BN2O21 (M+H)+ 1229.6760;
found 1229.6781.
BODIPY 3b. A solution of F-BODIPY 3 (9.17 mg, 0.028 mmol), 7b (26 mg, 0.028 mmol),
and trimethylsilyl chloride (75 µL, 0.57 mmol) in dry acetonitrile (2 mL) was heated under
microwave irradiation at 120 °C for 2 h. The solvent and excess volatile reagents were
removed at reduced pressure and the resulting residue was purified by flash chromatography
(CH2Cl2/MeOH 100:0 → 90:10) to afford 3b (25 mg, 73%) as an orange solid.
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1H NMR (CDCl3, 500 MHz): δ = 6.09 (1H, d, J = 2.3 Hz, H6´), 6.00 (1H, d, J = 2.3 Hz, H4´), 4.17
(2H, t, J = 5.3 Hz, H8´), 4.08 (2H, t, J = 4.6 Hz, H25´), 3.98 (2H, t, J = 5.3 Hz, H9´), 3.86-3.82 (4H,
m, H10´ and H26´), 3.72-3.70 (2H, m, H27´), 3.68-3.63 (48H, m, H11´-H22´ and H28´-H39´),
3.55-3.53 (4H, m, H23´ and H40´), 3.37 (6H, s, H24´ and H41´), 2.62 (3H, s, CH38), 2.35 (4H, q, J
= 7.5 Hz, CH3CH22 and CH3CH26), 2.33 (6H, s, CH31 and CH37), 2.12 (6H, s, CH33 and CH35),
0.97 (6H, t, J = 7.5 Hz, CH3CH22 and CH3CH26). 13C NMR (CDCl3, 125 MHz): δ = 164.6 (C5´),
163.5 (C3´), 162.2 (C7´), 161.2 (C1´), 152.4 (C3 and C5), 140.0 (C8), 137.0 (C7a and C8a),
133.0 (C1 and C7), 132.4 (C2 and C6), 99.9 (C2´), 94.7 (C4´), 93.5 (C6´), 72.1 (C23´ and C40´),
71.2 (C10´), 71.0 (C27´), 70.8, 70.7, 70.7, 70.7, 70.6 (from 70.8 to 70.6: C11´-C22´ and C28´-
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C39´), 69.6 (C26´), 69.4 (C9´), 69.1 (C8´), 67.5 (C25´), 59.2 (C24´´ and C41´´), 17.4 (CH38), 17.2
(CH3CH22 and CH3CH26), 15.1 (CH3CH22 and CH3CH26), 14.7 (CH31 and CH37), 13.0 (CH33 and
CH35). HRMS (API-ES+) calcd. for C59H101BN3O21 (M+NH4)+ 1198.7025; found 1198.7024.
BODIPY 2c. A suspension of F-BODIPY 2 (15 mg, 0.043 mmol), 7c (20 mg, 0.031 mmol),
and trimethylsilyl chloride (200 µL, 1.55 mmol) in dry acetonitrile (2 mL) was heated under
microwave irradiation at 50 °C for 40 minutes, at 80 °C for 30 minutes, at 100 °C for 30
minutes, and at 120 °C for 30 minutes. The solvent and excess volatile reagents were removed
at reduced pressure and the resulting residue was purified by reversed-phase column flash
chromatography (H2O/MeCN 100:0 → 0:100). The fractions containing product were
combined and lyophilized to afford 2c (16 mg, 53%) as a red solid.
4´
3´
2´
5´
6´
1´
7a
8
8a
7
1
6
2
N
O
N
B
5
3
O
1´´
3´´
4´´
O
2´´
7´´
5´´
O
6´´
8´´
O
15´´
16´´
9´´
O
O
17´´
10´´
11´´
18´´
N
N
12´´
19´´
14´´
20´´
13´´
O
21´´
S
O
S
O
O
O
O
1H NMR (CD3OD, 400 MHz): δ = 7.08 (2H, s, H3´ and H5´), 6.26 (1H, d, J = 1.8 Hz, H4´´/H6´´),
6.18 (1H, d, J =1.8 Hz, H6´´/H4´´), 6.10 (2H, s, H2 and H6), 4.25-4.24 (4H, m, H8´´ and H15´´),
4.15 (2H, br m, H10´´/H17´´), 4.01 (2H, br m, H17´´/H10´´), 3.97 (2H, t, J = 3.9 Hz, H16´´/H9´´),
3.89 (2H, t, J = 3.9 Hz , H9´´/H16´´), 3.65-3.58 (8H, m, H11´´, H12´´, H18´´and H19´´), 3.18 (6H,
28